Abstract Graft-versus-host disease (GvHD) is a serious complication following allogeneic hematopoietic cell transplantation and is characterized by the dysregulated immune response to allogeneic antigen stimulation after donor immune reconstitution, followed by the subsequent destruction of host tissues. Despite prophylactic measures, the incidence of acute GvHD remains substantially high. Primary prophylaxis and treatment relies on immunosuppression, elevating the risk of infection. New approaches are thus critically needed and biologic approaches hold the promise to help without immunosuppression. Regulatory T cells (Tregs) are key mediators of immunity and tolerance, and have been the recent focus in the study of GvHD, as well as a promising cellular therapy for reducing the risk of GvHD. An early phase clinical trial using ex vivo expanded Tregs demonstrated the safety of Treg infusion in GvHD patients; however, the efficacy for reducing the severity of GvHD was modest. Several factors, however, limit their clinical application. There is increasing evidence suggesting that optimal Treg function is impaired by inflammatory factors. Our preliminary studies have demonstrated a novel role for IL-27 in enhancing the suppressive function of Tregs in this context. In a murine model of T-cell mediated intestinal inflammation, we demonstrated that IL-27 signaling in Tregs is essential for Treg function in vivo. While wild-type Tregs are fully capable of suppressing T cell mediated intestinal inflammation, Tregs deficient in IL-27 receptors are unable to suppress inflammation. We further demonstrated that pre-stimulating Tregs with IL-27 significantly enhances their suppressive function in both intestinal inflammation and allogeneic GvHD. Based on these observations, we hypothesize that IL-27 pre- stimulation enhance Treg suppressive function and will have the capability of effectively preventing GvHD development. Two specific aims are proposed to address this hypothesis. Aim 1 will test the role of IL-27 signaling in Tregs in preventing GvHD in a murine allogeneic GvHD model. Aim 2 will test the therapeutic role of IL-27 pre-stimulation in human Tregs in a xenogenic model of GvHD. This study will generate proof-of- concept results for the development of highly efficacious IL-27 based Treg therapy for the prevention and attenuation of GvHD.